Genetically engineered insulin-producing cells, surrogate ?? cells, offer a solution to the shortage of ?? cells available for transplantation in individuals with Type 1 diabetes. Hepatocytes are a promising starting cell in the quest for surrogate ?? cells. However, achieving glucose-regulated insulin secretion in the genetically engineered liver cells is a difficult task as liver cells do not possess regulated secretory pathway. NeuroD, a pancreatic ?? cell transcription factor is involved in the differentiation of endocrine pancreatic cells and also directly regulates the expression of genes in the differentiated cells. However, little is known about the role of NeuroD in the glucose-regulated insulin secretion. The aim of this study was to investigate whether NeuroD induces glucose-regulated insulin secretion in the insulin-producing rat liver cell line, FAO-ins. The human NeuroD gene was stably expressed in FAO-ins cells. In the transfected cells (FAOins-Nd) the expression of genes encoding transcription factor Foxa2, L-type calcium channel subunits and secretory granule protein CgA was up-regulated. FAOins-Nd also showed greater intracellular insulin content and secretion as well as released insulin in a regulated manner to calcium stimulus. Further, growth factors namely betacellulin, activin A, nicotinamide and exendin-4 as well as insulin secretagogues such as theophylline, IBMX and carbachol were examined by static incubation in inducing glucose-regulated insulin secretion. Exendin-4 and insulin secretagogues stimulate insulin secretion in the presence of 1.5 mM glucose but the addition of 20 mM glucose had no further stimulatory effect. These results indicate that FAOins-Nd cells are sensitive to glucose and the release of insulin is non-glucose dependent. Overexpression of NeuroD and further treatment with exendin-4 or insulin secretagogues up-regulated insulin secretion but did not render these cells glucose-responsive. An attempt was made to generate transgenic NOD mice expressing large amounts of insulin in the liver using PEPCK promoter with SV40 poly adenylation sequence. Transgenic NOD mice were generated and the presence of insulin transgene was demonstrated. However, insulin mRNA and protein were not expressed in the liver of transgenic mice.
| Identifer | oai:union.ndltd.org:ADTP/187428 |
| Date | January 2007 |
| Creators | Appavoo, Mathiyalagan, Prince of Wales Clinical School, UNSW |
| Publisher | Awarded by:University of New South Wales. Prince of Wales Clinical School |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Copyright Mathiyalagan Appavoo, http://unsworks.unsw.edu.au/copyright |
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